The interaction between FUra/LV and recombinant human IFN-gamma is synergistic in colon carcinoma cells, dependent on FUra/LV-induced DNA damage, the Fas signaling pathway, and independent of p53. A Phase I trial of FUra/LV combined with IFN-gamma demonstrated responses in heavily pretreated and untreated patients. We propose to build on these studies with the long term objective to develop highly effective therapeutic strategies for the treatment of colon carcinoma. The mechanism of interaction between the FUra/LV, IFN-gamma and Fas signaling pathways involves alternative sites in addition to Fas. In HCT116 isogenic cell lines, p21Cip1 -/- cells, but not wild-type or p53 -/- cells, are resistant to the cytotoxic effects of TS inhibition and in contrast, are highly sensitive to IFN-gamma. P21Cip1 -/- cells accumulate in S-phase in contrast to wild-type cells, which progress through S-phase and undergo apoptosis. FUra/LV and IFN-gamma, upregulate the expression of caspases -3 and -8. In cells undergoing apoptosis, cleavage of caspase-3 occurs prior to caspase-8, independent of Bcl-2, suggesting that caspase-3 may be a direct target of drug interaction. We will test the hypothesis that p21Cip1 is a critical target of interaction between the signaling pathways for IFN-gamma and TS inhibitors, and that IFN-gamma is cytotoxic (p21Cip1 -/- cells) or a sensitizing agent (wild-type cells) due to regulation at the level of caspase-3. We have demonstrated that human colon carcinoma cell lines are highly sensitive to the cytotoxic ligand TRAIL in the presence of cycloheximide. By inhibiting PKC or CK2 survival pathways, cell lines also become highly TRAIL sensitive. We will test the hypothesis that inhibition of survival pathways involving phosphorylation can overcome resistance of colon carcinoma cells to TRAIL. IFN-gamma has demonstrated synergistic interaction when combined with both FUra/LV and/or TRAIL for all combinations examined. We will test the hypothesis that direct ligation of the TRAIL receptors can further sensitize colon carcinomas to IFN-gamma and to FUra/LV using cultured cell lines and xenograft models. It is anticipated that the proposed studies will identify critical targets that are convergent between death receptor, Fura/LV and IFN-gamma signaling pathways that determine synergistic interactions in colon carcinoma, and will further our knowledge in the development of highly effective therapeutic approaches.